Utilization of double-stranded RNA to inhibit gene expression in a sequence-specific manner has revolutionized the drug discovery industry. In mammals, RNA interference, or RNAi, is mediated by 19- to 29-nucleotide long, double-stranded RNA molecules referred to as small interfering RNAs that are derived by enzymatic cleavage of long, double-stranded RNA within cells in vivo. RNAi agents can be synthesized chemically or enzymatically outside of cells and subsequently delivered to cells (see, e.g., Fire, et al., Nature, 391:806-11 (1998); Tuschl, et al., Genes and Dev., 13:3191-97 (1999); and Elbashir, et al., Nature, 411:494-498 (2001)); or can be expressed in vivo by an appropriate vector in cells (see, e.g., McCaffrey, et al. Nature Biotech. 21(6): 639-644 (2003)).
However, in vivo delivery of unmodified RNAi as an effective therapeutic for use in humans faces a number of technical hurdles. First, due to cellular and serum nucleases, the half life of RNA injected in vivo is only about 70 seconds (see, e.g., Kurreck, Eur. J. Bioch. 270:1628-44 (2003)). Efforts have been made to increase stability of injected RNA by the use of chemical modifications; however, there are several occurrences in which chemical alterations led to increased cytotoxic effects. In one specific example, cells were intolerant to doses of an RNAi duplex in which every second phosphate was replaced by phosphorothioate (Harborth, et al., Antisense Nucleic Acid Drug Rev. 13(2): 83-105 (2003)). Other hurdles include providing tissue-specific delivery, as well as being able to deliver the RNAi agents in amounts sufficient to elicit a therapeutic response, but that are not toxic.
Several options are being explored for RNAi delivery, including the use of viral-based vector systems that can infect target cells, and deliver and express RNAi molecules in situ. Typically, small RNAs of approximately 70 nucleotides are transcribed as short hairpin precursors (shRNA) from a viral vector backbone. Once transcribed, the shRNA are processed by the enzyme Dicer into the appropriate active RNAi species. Viral-based delivery approaches attempt to exploit the targeting properties of viruses to generate tissue specificity and once appropriately targeted, rely upon the endogenous cellular machinery to generate sufficient levels of the RNAi species to achieve a therapeutically effective dose.
Currently, the most commonly used viruses for delivery of target sequences are those based upon systems evolved from retrovirus, herpes simplex virus (HSV) or adenovirus (Ad). All of these vectors can accommodate rather large inserts and can be produced in therapeutically relevant titers. However, in all systems, there are concerns relating to development of cancer (Cavazzana-Calvo, et al., Science, 288:669-72 (2000)), as well as undesirable host immune responses and resulting toxicity in patients. Another virus that is useful for delivering RNAi is adeno-associated virus (AAV).
One useful application of RNAi therapeutics is as an anti-viral agent. In general, RNA viruses depend on RNA/DNA-dependent RNA polymerase for replication. Such RNA/DNA polymerases replicate the viral genome with comparatively low fidelity, the functional consequence of which produces genomes with an exceptionally high number of mutations. This results in the ability of rapidly evolving progeny virions to evade common immunological and chemical antiviral agents. Thus, similar to the effects observed with small molecule therapeutics, the relative potency and efficacy of the RNAi therapeutic may decrease as a result of viral evolution during long term treatment. In one study, HIV escape mutants that contained a single nucleotide change appeared 35 days after delivery of an expressed shRNA (Boden, et al., J. Virol. 77(21): 11531-11535 (2003)). In another study, poliovirus escape mutants could be detected in as little as 54 hours post-infection in cells that had been transfected with pre-synthesized RNAi. Yet, the simultaneous delivery of two RNAis against multiple target sequences within the virus significantly delayed the onset of escape variants (see Gitlin, et al., Nature. 418: 430-434 (2002)).
Thus, there is a need in the art to develop stable, effective RNAi therapeutics. The present invention satisfies this need in the art.